Monitoring hybridization during polymerase chain reaction

The polymerase chain reaction (PCR) is usually analyzed by gel electrophoresis for size separation of PCR products. Additional separation techniques, such as single-stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and denaturing high-performance liquid chromatography (DHPLC), can also be used to scan for sequence alterations. These techniques are all based on the effect of PCR product hybridization on mobility. Hybridization can also be monitored with fluorescence during PCR without chromatographic or electrophoretic separation. Continuous monitoring of PCR allows the detection, quantification and sequence specificity of PCR products to be assessed, often without any need for further analysis. In such a closed system, PCR quantification with sensitivity to the single copy level can be achieved using either double-stranded DNA binding dyes or fluorescently labeled allele-specific oligonucleotide (ASO) probes. Melting curve analysis with ASO probes can be used to genotype various alleles, including single base alterations. The integration of rapid cycle PCR and ASO probes in an automated system greatly facilitates research and clinical applications of nucleic acid analysis in genetics, oncology, and infectious disease.